Factors driving the increase in drug-resistant tubercu-losis (TB) in the Eastern Cape Province, South Africa, are not understood. A convenience sample of 309 drug-sus-ceptible and 342 multidrug-resistant (MDR) TB isolates, collected July 2008–July 2009, were characterized by spoligotyping, DNA fingerprinting, insertion site mapping, and targeted DNA sequencing. Analysis of molecular-based data showed diverse genetic backgrounds among drug-sensitive and MDR TB sensu stricto isolates in con-trast to restricted genetic backgrounds among pre–exten-sively drug-resistant (pre-XDR) TB and XDR TB isolates. Second-line drug resistance was significantly associated with the atypical Beijing genotype. DNA fingerprinting and sequencing demonstrated that the pre-XDR and XDR atypical Beijing isolates evolved from a common progeni-tor; 85% and 92%, respectively, were clustered, indicating transmission. Ninety-three percent of atypical XDR Beijing isolates had mutations that confer resistance to 10 anti-TB drugs, and some isolates also were resistant to para-ami-nosalicylic acid. These findings suggest the emergence of totally drug-resistant TB.
T
he emergence of drug-resistant tuberculosis (TB) is of major concern to TB control in South Africa.A countrywide survey in 2002 revealed that 1.8% of all new TB patients and 6.7% of TB patients who had under-gone previous treatment had multidrug-resistant (MDR) TB (resistant to at least isoniazid and rifampin) (1). This finding translates to an estimated annual case load of 13,000 MDR TB cases, placing South Africa fourth among countries where MDR TB is highly prevalent (1). How-ever, this number may be an underestimation; 2 recent studies (2,3) suggested that the proportion of MDR TB cases may be substantially higher than the World Health Organization (WHO) estimate (3). In addition, only 4,143 of the 9,070 patients (46%) who received a diagnosis of MDR TB in 2009 received treatment, possibly because of resource constraints, creating a situation in which control was bound to fail (4). This conclusion is supported by the diagnosis of 594 extensively drug- resistant (XDR) TB cases (MDR plus additional resistance to a fluoroquino-lone and any second-line injectable drug) in that year (4). The cure rate of patients with drug-resistant TB is <50% for those with MDR TB (5), whereas culture conversion was observed in only 19% of XDR TB case-patients dur-ing the follow-up period, irrespective of HIV status (6).
Most cases of MDR TB and XDR TB in South Af-rica have been detected in KwaZulu-Natal, Western Cape, and Eastern Cape Provinces (4). Statistics from the Eastern Cape showed the largest increase in the number of MDR TB cases, rising from 836 cases in 2006 to 1,858 cases in 2009 (2.2 fold increase) (4). The reason for this dramatic increase in MDR TB cases remains to be determined.
Molecular epidemiologic data from the neighboring Western Cape Province have demonstrated that MDR TB is spread by primary transmission (7–9), which accounts for nearly 80% of reported MDR TB cases (2). To date, only 1 molecular epidemiologic study has been reported for the
of Extensively and Totally
Drug-Resistant Tuberculosis,
South Africa
Marisa Klopper, Robin Mark Warren, Cindy Hayes, Nicolaas Claudius Gey van Pittius, Elizabeth Maria Streicher, Borna Müller, Frederick Adriaan Sirgel, Mamisa Chabula-Nxiweni,
Ebrahim Hoosain, Gerrit Coetzee, Paul David van Helden, Thomas Calldo Victor, and André Phillip Trollip
Author affiliations: Stellenbosch University, Cape Town, South Af-rica (M. Klopper, R.M. Warren, N.C. Gey van Pittius, E.M. Streich-er, B. MüllStreich-er, F.A. Sirgel, P.D. van Helden, T.C. Victor, AP.. Trollip); National Health Laboratory Services, Port Elizabeth, South Africa (C. Hayes, G. Coetzee); Swiss Tropical and Public Health Institute, Basel, Switzerland (B. Müller); and University of Basel, Basel (B. Müller); Nelson Mandela Metropolitan Municipality, Port Elizabeth (M. Chabula-Nxiweni); and Human Sciences Research Council, Port Elizabeth (E. Hoosain)
Eastern Cape (10), and it showed that 50% of rifampin-re-sistant TB isolates (including MDR TB isolates) belonged to the Beijing genotype and that “atypical” Beijing strains were significantly overrepresented. These strains harbored rare mutations in the inhA gene promoter (G-17A) and rpoB gene (GAC→GTC nucleotide substitutions in codon 516), which have previously been associated with a high fitness cost (11). The authors demonstrated that the spread of these strains was facilitated by HIV co-infection, thereby raising concern for the spread of drug-resistant strains in vulnerable populations (10).
A recent epidemiologic study conducted in the Eastern Cape estimated that 75.6% of XDR TB cases with complete data were a result of ongoing transmission (12). Treatment outcomes were dismal; 58% of case-patients died within 1 year, and culture conversion was observed in only 8.4% of case-patients after 143 days of treatment (12), raising concern that these patients had an untreatable form of TB. This situation is similar to the Tugela Ferry outbreak in KwaZulu-Natal Province (13), which highlighted the need for improved basic control measures, including rapid diag-nostics and infection control methods (14).
This study aimed to describe the Mycobacterium tu-berculosis strain population structure among MDR TB and XDR TB case-patients in Eastern Cape Province, South Af-rica, in order to determine whether the epidemic was driven by acquisition or transmission of resistance and to describe the extent of resistance within these strains. These findings will inform TB control efforts to better implement mea-sures to curb emergence or the spread of drug-resistance.
Materials and Methods
Study Population
Sputum specimens were collected from persons at high-risk for suspected TB (previously treated case-pa-tients and close contacts of known pacase-pa-tients with drug- resis-tant cases) in accordance with the National TB Control Pro-gram. Specimens that were collected at healthcare facilities in the Eastern Cape Province were submitted to the Na-tional Health Laboratory Service (NHLS) in Port Elizabeth for TB drug susceptibility testing (DST). From July 2008 through July 2009, a convenience sample of sputum cul-tures, shown to be either fully drug-susceptible or resistant to at least isoniazid and rifampin (MDR TB) by the NHLS, was submitted to Stellenbosch University in Cape Town for subsequent genotyping. Only limited demographic and clinical data were available for each patient: a unique identifier (assigned by the NHLS), the date sputum was obtained, the name of the clinic/hospital where the sample originated, and the routine DST pattern. The unique identi-fier was used to identify the first available isolate from 309 drug-susceptible and 342 MDR TB case-patients included
in the study. This study was approved by the ethics com-mittee of Stellenbosch University, Faculty of Health Sci-ences (N09/11/296).
Drug Susceptibility Testing
Sputum samples were processed by the NHLS for rou-tine TB diagnosis by smear microscopy and culture. Each sputum specimen was decontaminated by using the stan-dard N-acetyl-L-cysteine-sodium hydroxide method and cultured in mycobacteria growth indicator tube (MGIT) 960 medium until a positive growth index was observed. DST was done by the indirect proportion method with the BACTEC MGIT 960 system (BD Bioscience, Sparks, MD, USA), according to the manufacturer’s instructions. We initially tested resistance against isoniazid and rifampin, followed by testing for resistance against streptomycin and ethambutol if the isolate was resistant to either isoniazid or rifampin. Second-line DST was done in 7H10 medium containing 2 µg/mL of ofloxacin, 4 µg/mL of amikacin, or 5 µg/mL of ethionamide. DST for para-aminosalicylic acid was done at Stellenbosch University in MGIT 960 medium containing 4.0 µg/mL, 8 µg/mL, and 16 µg/mL of para-aminosalicylic acid (15).
Molecular-based Analysis
Crude DNA was prepared by boiling a 200-µL aliquot of a mycobacteria-positive MGIT culture, and this was used as a template for subsequent PCR analysis (16). Each isolate was spoligotyped by using the international stan-dardized method (17) and grouped into genotypes accord-ing to previously described spoligotype signatures (18). Beijing genotype strains were subclassified as either “typi-cal” or “atypical,” according to the presence or absence of an IS6110 insertion in the noise transfer function (NTF) region (19,20). The atypical Beijing genotype strains were further classified by using the international standardized IS6110 DNA fingerprinting method (21). For atypical Beijing strains that were drug-sensitive according to DST, sensitivity to isoniazid and rifampin was confirmed by se-quencing the katG and rpoB genes. In MDR atypical Bei-jing strains, mutations conferring resistance to isoniazid, rifampin, ethambutol, pyrazinamide, ofloxacin, streptomy-cin, amikastreptomy-cin, kanamystreptomy-cin, and capreomycin were identi-fied by sequencing the inhA promoter and the katG, rpoB, embB, pncA, gyrA, and rrs genes, respectively (22,23). Iso-lates were grouped as as follows: MDR TB sensu stricto (MDR TB ss, that is, MDR strains excluding identified pre-XDR, MDR plus additional resistance to either a fluoro-quinolone or any second-line injectable anti-TB drug) [24] and XDR strains); pre-XDR TB; or XDR TB, according to high confidence mutations. This method of grouping was selected because routine DST was not done for all of the anti-TB drugs on all of the isolates. Furthermore, a poor
correlation was observed between high-confidence muta-tions and routine second-line DST. Isolates were consid-ered to belong to the same cluster (implying ongoing trans-mission) if identical mutations were observed in all of the genes sequenced.
Results
A convenience sample of 309 drug-sensitive and 342 MDR TB isolates from patients from Eastern Cape Province was collected during the study period. These isolates were submitted to Stellenbosch University for molecular-based analysis. Analysis of the population structure of these iso-lates by spoligotyping identified 52 and 29 different spoligo-type patterns among drug-sensitive and MDR TB strains, re-spectively. Among drug-sensitive and MDR isolates, 22/52 and 14/29 spoligotype patterns were previously recorded in the fourth international spoligotyping (SpolDB4) database. These represented 275 (89.0%) of 309 drug-sensitive iso-lates and 327 (95.6%) of 342 MDR isoiso-lates. Notably, 84% of MDR isolates constituted only 3 different spoligotypes (Table 1), namely, Beijing, LAM3, and LAM4. These find-ings indicate transmission of these strains.
Table 1 shows the classification of spoligotypes, ac-cording to the degree of drug resistance, in which drug resistance is expressed as the result of culture-based or based DST. In this study, we used molecular-based DST to define the extent of drug-resistance in rou-tinely diagnosed MDR TB isolates. Accordingly, 119 (38.5%) of the drug-susceptible isolates and 236 (69.0%) of the MDR TB isolates were of the Beijing genotype. Subclassification of Beijing genotype strains showed that 11(9.2%) of 119 drug-sensitive and 217 (91.9%) of 236 MDR strains belonged to the “atypical” subgroup of
the Beijing genotype, as indicated by the absence of an IS6110 element in the NTF region.
Analysis of mutations conferring resistance to first- and second-line anti-TB drugs enabled grouping of the MDR isolates: 136 MDR ss, 98 pre-XDR, and 108 XDR. Using these groupings, we found that isolates with a higher degree of resistance were more likely to have an atypical Beijing genotype (drug sensitive: 11/309 [3.6%, 95% CI 1.8%– 6.3%], MDR ss: 29/136 [21.3%, 95% CI 14.8%–29.2%] vs. pre-XDR: 85/98 [86.7%, 95% CI 78.4%–92.7%] vs. XDR: 103/108 [95.4%, 95% CI 89.5%–98.5%]).
We analyzed DNA sequencing data for the first avail-able isolate from each patient infected with an MDR atypi-cal Beijing strain (n = 217) and performed IS6110 finger-printing for a subset of these isolates (n = 110) to establish whether the overabundance of the atypical Beijing genotype among patients with pXDR TB and XDR TB strains re-flected ongoing transmission. IS6110 DNA fingerprinting showed that all of these patients were infected with closely related atypical Beijing strains with only minor differences in the banding patterns (online Technical Appendix, Figures 1, 2, wwwnc.cdc.gov/EID/article/19/3/12-0246-Techapp1. pdf), thereby suggesting clonal dissemination.
The online Technical Appendix Table shows that 216 (99.5%) of 217 of the MDR atypical Beijing genotype strains harbored an identical katG (AGC315ACC) muta-tion, whereas 209 (94.9%) of 217 had a distinctive rrs (A513C) gene mutation. This finding suggests that these mutations were acquired before dissemination. Subse-quently, resistance to rifampin, ethambutol, pyrazinamide, amikacin, and ofloxacin was acquired in various combi-nations. Of the 29 atypical Beijing MDR ss isolates, 22 (75.9%) were grouped into 4 clusters according to mutations Table 1. Spoligotype classification of drug-sensitive and MDR TB isolates, Eastern Cape Province, South Africa, 2008–2009*
Spoligotype
family† ST no. Sensitive Culture-based DST, no. (%) MDRss Pre-XDR XDR MDRss Molecular-based DST, no. (%) Pre-XDR XDR Atypical Beijing 1 11 (3.6) 41 (27.0) 98 (92.5) 78 (92.9) 29 (22.5) 85 (87.6) 103 (95.4) Typical Beijing 1 108 (35.0) 19 (12.5) 0 0 18 (14.0) 1 (1.0) 0 H 36; 47; 50; 62 7 (2.3) 2 (1.3) 1 (0.9) 0 2 (1.6) 1 (1.0) 0 LAM3 33; 130; 211 66 (21.4) 12 (7.9) 2 (1.9) 0 12 (9.3) 0 (0) 0 LAM4 60; 811 6 (1.9) 32 (21.1) 4 (3.8) 2 (1.9) 29 (22.5) 3 (3.1) 2 (1.9) LAM (other) 4; 20; 42; 398 7 (2.3) 1 (0.7) 0 1 (1.2) 1 (0.8) 0 1 (0.9) MANU2 1247 0 0 0 2 (2.4) 1 (0.8) 0 1 (0.9) S 34; 71 8 (2.6) 8 (5.3) 0 1 (1.2) 8 (6.2) 0 1 (0.9) T 44; 53; 73; 254; 926; 1240 51 (16.5) 18 (11.8) 0 0 13 (10.1) 5 (5.2) 0 U 443; 519; 790 1 (0.3) 2 (1.3) 0 0 2 (1.6) 0 0 X 18; 92; 119; 1751 6 (1.9) 3 (2.0) 0 0 3 (2.3) 0 0 CAS 21; 26; 1092 4 (1.3) 0 0 0 0 0 0
Orphan Not assigned 34 (11.0) 14 (9.2) 1 (0.9) 0 11 (8.5) 2 (2.1) 0
Total 309 152 106 84 129 97 108
Total MDR 342 334‡
*MDR TB, multidrug-resistant tuberculosis; ST, shared type (17); DST, drug susceptibility testing; MDRss, MDR sensu stricto; Pre-XDR, pre–extensively drug resistant; XDR, extensively drug resistant.
†For Beijing isolates a distinction was made between typical and atypical based on the presence or absence of an IS6110 insertion in the noise transfer region (18,19).
(mutation pattern [MP]) in the inhA promoter and the katG, rpoB, embB, pncA, rrs, and gyrA genes (cluster size ranged from 3 to 12 cases; online Technical Appendix Table: MP2, MP17, MP32, MP34), whereas 7 had unique MPs (online Technical Appendix Table 2: MP23, MP25, MP30, MP31, MP41, MP44, MP48). Similarly, the 85 atypical pre-XDR Beijing isolates showed 11 different MPs, of which 81 (95.3%) were grouped into 7 clusters (cluster size ranged from 2 to 62 cases; online Technical Appendix Table: MP3, MP5, MP18, MP26, MP28, MP35, MP38). The genotype of the largest pre-XDR TB cluster was characterized by an inhA promoter mutation at position 17 and the katG AG-C315ACC, rpoB GAC516GTC, embB ATG306ATA, rrs A513C, and rrs A1401G nucleotide substitutions as well as an insertion in the pncA gene at position 172G. This MP was characteristic of 81 (78.6%) of 103 of the atypical Beijing XDR TB isolates and, for ease of reference, will be called MP5 (online Technical Appendix Table). By contrast, only 3 of the 29 atypical Beijing MDR ss isolates showed the same mutation pattern for these genes, excluding the rrsA1401G mutation (MP2). Ten different atypical XDR Beijing MPs emerged from the MP5 progenitor by mutation in the gyrA gene. Of these, 6 MPs showed clustering (cluster size ranged from 2 to 46 cases, MP6–11), and 4 had unique mutations conferring ofloxacin resistance (MP12–16). Clus-tering of both the pre-XDR and XDR genotypes suggests transmission after the acquisition of additional resistance. Of the remaining 22 atypical XDR Beijing isolates, 12 distinct resistance MPs were observed, of which 11 isolates were clustered (MP27) and 11 had unique genotypes (MP19–22, MP24, MP29, MP39–40, MP42–43, MP47).
Spatial analysis of the patients’ origins showed that pre-XDR and XDR isolates with an atypical Beijing geno-type were found in 5 of 8 district municipalities (Figure; online Technical Appendix Table). The largest atypical pre-XDR Beijing genotype cluster (MP5) was identified in 4 adjacent district municipalities (online Technical Ap-pendix Table), and the largest XDR TB cluster (MP6) was identified in 3 of these districts as well as in an ad-ditional district, which suggests the past spread of these genotypes.
The presence of mutations in target genes known to confer resistance with high confidence indicated that 95.1% (98/103) of the atypical XDR Beijing isolates were resis-tant to at least 10 anti-TB drugs: isoniazid, rifampin, eth-ambutol, pyrazinamide, streptomycin, amikacin, kanamy-cin, capreomykanamy-cin, ethionamide, and ofloxacin. The extent of drug resistance in these isolates was underestimated by routine DST (Table 2). The correlation between molecular-based drug-resistance and routine culture-molecular-based DST was 99.6% for isoniazid, 100% for rifampin, 28% for etham-butol, 92% for streptomycin, 93% for amikacin, 27% for capreomycin, 52% for ethionamide, and 86% for ofloxacin (Table 2). Routine DST for pyrazinamide, kanamycin, cy-closerine, and para-aminosalicylic acid was not performed. DST for para-aminosalicylic acid was done at Stellenbosch University on 45 isolates; 9 showed resistance at a level of >4.0 µg/mL.
Discussion
Review of routine DST results highlights the severity of the drug-resistant TB epidemic in South Africa (4) and thereby emphasizes the urgent need for curbing the rising incidence of drug resistance in the country. This result can only be achieved by implementing appropriate intervention strategies based on knowledge of the mechanisms fueling this epidemic. Recently, molecular epidemiologic tech-niques were used in combination with classical epidemio-logic data to enhance understanding of the TB epidemic in different settings. Those studies have quantified the rela-tive proportion of acquisition versus transmission and have described the population structure of M. tuberculosis over time (7,10,22,24,25). Using these approaches, we show that patients with MDR TB in the Eastern Cape could be di-vided into 2 distinct groups: isolates from patients infected with MDR ss showed diverse genetic backgrounds, while isolates from patients infected with pre-XDR TB and XDR TB showed restricted genetic backgrounds.
The finding that the pre-XDR TB and XDR TB strains are genetically distinct when compared to the MDR ss strains is counterintuitive because we would expect all MDR TB strains to have had an equal chance of acquiring Table 2. Correlation of culture-based and molecular-based drug-susceptibility testing among atypical Beijing isolates, South Africa, 2008–2009*
Drug/gene CB-DST R, MB-DST R CB-DST S, MB-DST S CB-DST R, MB-DST S CB-DST S, MB-DST R Total Correlation, %
INH/katG 217 9 1 0 227 99.6 RIF/rpoB 219 9 0 0 228 100 STR/rrs500 191 6 2 16 215 91.6 EMB/embB 56 5 2 152 215 28.4 ETH/inhA promoter 76 25 5 86 192 52.6 OFL/gyrA 78 93 0 29 200 85.5 AMK/rrs1400 167 32 7 9 215 92.6 CAP/rrs1400 21 38 1 155 215 27.4
*CB-DST, culture-based drug susceptibility testing; R, resistant; MB-DST, molecular-based DST; S, sensitive; INH, isoniazid; RIF, rifampin; STR, streptomycin; EMB, ethambutol; ETH, ethionamide; OFL, ofloxacin; AMK, amikacin; CAP, capreomycin.
resistance to second-line anti-TB drugs. The absence of second-line resistance among a large number of different MDR TB genotypes suggests that under the current MDR TB treatment regimen, acquisition of additional resistance in MDR ss strains is reduced. Conversely, analysis of the DNA sequencing data showed a significant association be-tween the atypical Beijing genotype and mutations con-ferring second-line resistance. This demonstrates that this genotype has acquired resistance to the level of pre-XDR TB, which in turn has spread and thereafter has acquired additional resistance to the level of XDR TB, followed again by transmission. An alternative explanation would be that the atypical Beijing genotype acquires resistance by conferring mutations more readily than other geno-types. However, the convergent evolution of 7 different mutations within a single genotype is highly unlikely.
Analysis of the locations of the pre-XDR TB case-pa-tients infected with this clone shows that it had a wide geo-graphic distribution, which suggests that this genotype has been in circulation for an extended period. This conclusion was further supported by the analysis of the evolutionary order in which resistance was acquired (online Technical Appendix Table), which showed that the ancestral clone first acquired resistance to isoniazid and streptomycin. This could be explained by the treatment regimen used in the early 1960s, which was based on the combination of isonia-zid and streptomycin (26). A similar conclusion was drawn from whole genome sequence data which predicted that mu-tations conferring resistance to isoniazid and streptomycin were deeply rooted in the atypical Beijing genotype (27).
Given the extent of resistance in pre-XDR TB strains and the extremely limited treatment options available, the emergence of ofloxacin resistance was inevitable. This idea was supported by our molecular-based analysis of the XDR TB isolates, which demonstrated that resistance to a fluo-roquinolone had been acquired independently on several different occasions (several different gyrA mutations were observed), followed by amplification through transmission (clustering of XDR phenotypes was observed). However, the true extent of acquisition may be higher than predicted, given that the XDR TB isolates were cultured from samples from patients who resided in different district municipali-ties, and contact was unlikely because of the long distances. We suggest that the absence of routine second-line drug susceptibility testing and the treatment of MDR TB with an inadequate standardized regimen, according to the 2004 guidelines (www.sahealthinfo.org/tb/mdrtbguidelines.pdf) (6 months’ intensive phase: kanamycin, ethionamide, pyra-zinamide, ofloxacin, and cycloserine or ethambutol; 12–18 months continuation phase: ethionamide, ofloxacin, and cy-closerine or ethambutol) (28) may have led to the inappro-priate treatment of undiagnosed pre-XDR TB cases. This regimen would have prolonged the period of infectiousness
leading to transmission to close contacts and increased the risk of amplification of resistance (28,29). This problem has been recently addressed with the implementation of a re-vised treatment regimen (28) as well as routine second-line DST, which is now done on all isolates shown to be re-sistant to rifampin. However, these tests are culture-based, which exacerbates diagnostic delay and possible transmis-sion. This situation can be partially resolved with the imple-mentation of a genetic-based second-line drug susceptibil-ity test (29). However, the extent of resistance associated with the atypical Beijing genotype makes treatment options extremely difficult as these isolates are resistant to all first-line anti-TB drugs (isoniazid, rifampin, ethambutol, pyra-zinamide and streptomycin) and many of the second-line drugs (amikacin, kanamycin, ofloxacin, ethionamide, cap-reomycin). A limited number of isolates were also resistant to para-aminosalicylic acid. This suggests that the atypical Beijing genotype clone is evolving toward total drug resis-tance (defined as in vitro resisresis-tance to all first-line drugs, as well as aminoglycosides, cyclic polypeptides, fluoroquino-lones, thioamides, serine analogs, and salicylic acid deriva-tives [30]) with acknowledgment of WHO’s concern over the definition (31). Our molecular-based results are in ac-cordance with a recent study from the Eastern Cape, which documented extremely poor treatment outcomes for XDR TB case-patients (12). The authors found that these patients experienced a high death rate (58.4%) and low culture-con-version rates (8.4%) over a follow-up period of 143 days. They concluded that only 1.7 drugs per patient could be regarded as “effective” on the basis of DST results, previ-ous treatment records, or both. Given that this study was conducted concurrently with ours, it is highly likely that a large proportion of their patients were also infected with XDR TB strains with an atypical Beijing genotype. Thus, the poor treatment outcome may be related to the extent of
Figure. District municipalities in the Eastern Cape Province, South Africa. Map courtesy of F. W. van Zyl.
drug-resistance; however, we cannot exclude the possibility that the atypical Beijing genotype contributes to illness and death. A further concern is the knowledge that this clone is now spreading to other provinces in South Africa, pos-sibly due to migration. In Western Cape Province, an esti-mated 55% of XDR TB case-patients harbor isolates with the atypical Beijing genotype (32).
We acknowledge that this study has several limita-tions. First, clinical data were not available for this study, and thus it was not possible to establish the effects of drug resistance on treatment outcome. However, we do not be-lieve that the strains reported by Kvasnovsky et al. (12) differ from those analyzed in this study because the stud-ies were conducted concurrently. Second, our analysis of a convenience sample may have led to an overestimation of the proportion of pre-XDR TB and XDR TB cases in Eastern Cape Province. Third, our use of mutational data to categorize patient isolates as MDR ss, pre-XDR, and XDR is not the accepted standard. However, genetic DST has been endorsed by WHO for first-line anti-TB drugs, and mounting evidence indicates that high confi-dence mutations accurately predict second-line drug re-sistance (33).
The diagnostic dilemma facing TB control managers in Eastern Cape Province is how to rapidly identify case-patients at risk of harboring the atypical Beijing genotype to prioritize DST, ensure patient isolation, and adminis-ter appropriate treatment. Previous studies have shown a strong association between inhA promoter mutations and pre-XDR TB and XDR TB (34). Given that the Geno-type MTBDRplus test (35) has been implemented as the diagnostic standard in most NHLS laboratories in South Africa, we propose that this test could be used as a rapid screening tool to identify patients harboring drug-resis-tant atypical Beijing strains (34). To contain the spread of this virtually untreatable form of TB, control managers must make use of this information.
Acknowledgments
We thank the South African National Research Founda-tion, Harry Crossley FoundaFounda-tion, Wellcome Trust (grant WT-087383MA), and the International Atomic Energy Agency, TBAdapt (project no. 037919), for funding. We also thank Marianna De Kock, Claudia Spies, and Ruzayda van Aarde for technical support.
Ms Klopper is a PhD student in the Department of Biomedi-cal Sciences, Division of Molecular Biology and Human Genet-ics, Stellenbosch University, South Africa. She has been working in the drug-resistant TB group within the division, focusing on describing the drug-resistant TB epidemic in the Eastern Cape Province, with an emphasis on molecular aspects of the popula-tion structure of this epidemic.
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Address for correspondence: Robin Mark Warren, Centre of Excellence for Biomedical Tuberculosis Research/MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Faculty of Health Sciences, Stellenbosch University, PO Box 19063, Tygerberg, South Africa, 7505; email: rw1@sun.ac.za
